Abstract: The long-term objective of the proposed work is to develop Herpesvirus-derived vectors for anti-cancer immunotherapeutic applications. lmmunotherapy, the targeted alteration of immunological parameters to achieve a therapeutic outcome, has revolutionized the treatment of cancer. lmmunotherapeutic approaches to the treatment of cancer include vaccination, oncolytic virotherapy, chimeric antigen receptor T-cells (CAR-T), and checkpoint inhibitors. While these approaches have met with great success there are still many drawbacks to current immunotherapeutic approaches that include serious side effects seen with CAR-T and checkpoint inhibitors. A current focus of immunotherapy is on altering the immunosuppressive tumor microenvironment (TME). Changes in the TME are not only important to the establishment and maintenance of tumors but also mediate the development of resistance to chemical and physical tumor therapies. Our central hypothesis is that disruption of the immunosuppressive tumor microenvironment is an important mechanism of action for oncolytic herpesviruses. This hypothesis will be tested using three specific aims. Specific aim 1 seeks to understand how oncolytic virotherapy affects the tumor microenvironment. Specifically, we will assess quantitative, spatial and functional changes in both the lymphoid and myeloid components of the tumor microenvironment. This will be accomplished by assaying the phenotypic, transcriptional and functional changes in the myeloid and lymphoid cells during oncolytic virotherapy. For Specific aim 2 we will assess the development of antitumor specific T-cells during oncolytic virotherapy. The kinetics and magnitude of T-cell responses will be determined using next-generation sequencing, adoptive transfer experiments and functional assays. Further, in Specific aim 2b we will design recombinant oncolytic herpesviruses that express tumor associated antigens in a B 16F1 O murine melanoma model. Using these viruses we will determine how the delivery of tumor specific antigens by oncolytic viruses can promote enhanced tumor-directed T-cell responses. Finally, in aim 3 we will determine how previous exposure to HSV-1 affects HSV-1 oncolytic virotherapy. For this aim we will assess changes in the tumor microenvironment as well as tumor specific T-cell responses during oncolytic virotherapy in mice that are previously exposed to HSV-1 infection.